PROJECT 2 (Godwin) PROJECT SUMMARY The lack of scar-free healing and regeneration in humans imposes severe limitations on functional recovery after major traumatic injury, surgical interventions and disease. Default wound-repair in most adult human tissues initiates the formation of a protein/carbohydrate fibrotic network that progressively matures into dysfunctional scar tissue. In organs like the heart, lung and liver, formation of scar tissue can be deadly and contributes to ~45% of all U.S. deaths. Development of therapies that activate regeneration and scar-free repair programs in humans will transform modern healthcare. In contrast to humans and most other mammals, salamanders are capable of scar-free regeneration of almost all complex tissues including the limbs, heart, brain and spinal cord. Using the salamander as a model system, we recently demonstrated for the first time the critical role of the innate immune system in regulating scar-free regeneration. Specifically, we showed 1) that early infiltration of macrophages into damaged limb or heart tissue actively suppresses fibrosis and 2) that suppression of fibrosis is an essential step required for normal regeneration to occur. The cellular and molecular mechanisms by which macrophages suppress fibrosis after injury are completely unknown. The overarching goal of this proposal is to begin defining these mechanisms for the first time using salamander limb regeneration as a model system. Development of therapies that suppress fibrosis by modulating macrophage function may allow scar-free repair and regeneration of damaged tissues in humans. Our proposal will be the first to define how salamander macrophages suppress myofibroblast induction and scar tissue formation. We will use lineage tracing methods to identify the source of scar producing cells, which are the cellular targets of anti-fibrotic macrophage signaling. We will then define the macrophage subtypes that inhibit myofibroblast induction and fibrotic activation signals. Using RNA-sequencing, we will characterize gene expression patterns in macrophages that suppress myofibroblast induction. These studies will provide the first mechanistic insights into anti-fibrotic signaling pathways that allow macrophages to support scar-free healing. Finally, we will test the hypothesis that permanent scar tissue formation may be reversible by inhibition of lysyl oxidase, an enzyme that mediates crosslinking of the extracellular matrix, which, in turn, prevents scar-free healing and regeneration. This proposal is both significant and innovative as it will address a critical gap in our understanding of how fibrosis and scarring is overcome in a highly regenerative animal model. These new insights will form an essential step in the development of anti-fibrosis and pro-regenerative therapies for patients. !